Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria

ABSTRACT

The present invention relates generally to detection of antibiotic-resistant bacteria in a sample. In particular, the invention provides methods, compositions and kits for detecting and analyzing methicillin-resistant  Staphylococcus aureus  (MRSA) and other methicillin-resistant bacteria in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application of U.S.application Ser. No. 13/038,035, filed Mar. 1, 2011, which is acontinuation application of U.S. application Ser. No. 12/106,137 filedApr. 18, 2008, now U.S. Pat. No. 8,107,337, issued on Sep. 13, 2011,claims the benefit of the filing date of U.S. Provisional PatentApplication Ser. No. 60/907,848, filed Apr. 19, 2007. All applicationsto which priority is claimed are hereby incorporated by reference intheir entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to detection ofantibiotic-resistant bacteria in a sample. In particular, the inventionprovides methods, compositions and kits for detecting and analyzingmethicillin-resistant Staphylococcus aureus (MRSA) and othermethicillin-resistant bacteria in a sample.

BACKGROUND OF THE INVENTION

New strains and species of antibiotic-resistant bacteria are becomingincreasingly common in hospitals and other health-care facilities.Treatment options for infections caused by such bacteria are oftenlimited to costly medications that produce undesirable side effects.Antibiotic resistance has been detected in strains of a number ofbacteria, including Staphylococcus aureus, Oerskovia turbata,Aracanobacterium haemolyticum, Streptococcus bovis, Streptococcusgallolyticus, Streptococcus lutetiensis, Bacillus circulans,Paenibacillus, Rhodococcus, Enterococcus, Klebsiella, as well asanaerobic bacteria belonging to the Clostridium genus and Eggerthellalenta, and many other pathogenic bacteria.

Methicillin-resistant Staphylococcus aureus (MRSA) is one example of atype of antibiotic-resistant bacteria emerging as a majorepidemiological problem in hospitals throughout the world. MRSA strainsare resistant to beta-lactams including penicillins, cephalosporins,carbapenems, and monobactams, which are the most commonly usedantibiotics to cure S. aureus infections. Thus, MRSA infections can onlybe treated with toxic and costly antibiotics (such as Vancomycin andLinezolid), which, due in large part to their negative side effects, arenormally only used as a last line of defense. A recent development inMRSA evolution is the emergence of strains that are at least partiallyresistant to such last-line antibiotics. If these partially resistantstrains become fully resistant, there will be no effective treatment forinfections caused by those strains.

Early detection and treatment are the primary tools for mitigating thetransmission of constantly evolving strains of antibiotic-resistantbacteria. Traditional methods of screening for such bacteria require atleast 2-4 days for results, during which time the infection has ampleopportunity to spread. Rapid and accurate methods, compositions and kitsfor the detection of antibiotic-resistant bacteria are thereforeessential for minimizing their transmission and the pace of theirevolution.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods, compositions andkits for rapid detection of antibiotic-resistant bacteria, including inone non-limiting embodiment, methicillin-resistant bacteria.

In one aspect, the invention provides a method of detectingmethicillin-resistant S. aureus (MRSA) in a sample. In this aspect, themethod includes the steps: (i) providing a first set of primers wherethe primers are complementary to at least a portion of a mecApolynucleotide sequence; (ii) providing a second set of primers, wherethe primers are complementary to at least a portion of a bridgingregion; (iii) providing a third set of primers, where the primers arecomplementary to at least a portion of an S. aureus-specificpolynucleotide sequence, and where the S. aureus-specific polynucleotidesequence is not an orfX polynucleotide; (iv) combining the first, secondand third set of primers with the sample in a reaction mixture; (v)performing a multi-cycle amplification reaction with the reactionmixture; and (vi) determining cycle numbers of appearance of each of themecA, bridging region and S. aureus-specific polynucleotide sequences.In this aspect, the cycle numbers indicate whether MRSA is present in asample.

In another aspect, the invention provides a method of detectingmethicillin-resistant S. aureus (MRSA) in a sample. In this aspect, themethod includes the steps: (i) determining whether a mecA polynucleotideis present in the sample; (ii) determining whether a bridging regionpolynucleotide is present in the sample; and (iii) determining whetheran S. aureus-specific polynucleotide is present in the sample. In thisaspect, the S. aureus-specific polynucleotide is not an orfXpolynucleotide. In this aspect, if the mecA polynucleotide, the bridgingregion polynucleotide, and the S. aureus-specific polynucleotide are allpresent in said sample, then MRSA is present in said sample.

In still another aspect, the invention provides a method of detectingmethicillin-resistant S. aureus (MRSA) in a sample. In this aspect, themethod includes the steps: (i) providing a first set of primers, wherethe first set of primers are complementary to at least a portion of amecA polynucleotide sequence; (ii) providing a second set of primers,where the second set of primers are complementary to at least a portionof an MSSA-orfX polynucleotide sequence; (iii) providing a third set ofprimers, where the third set of primers are complementary to at least aportion of an S. aureus-specific polynucleotide sequence, and where theS. aureus-specific polynucleotide sequence is not an orfXpolynucleotide; (iv) combining the first, second and third set ofprimers with the sample in a reaction mixture; (v) performing amulti-cycle amplification reaction with said reaction mixture; and (vi)determining cycle numbers of appearance of each of the mecA, MSSA-orfXand S. aureus-specific polynucleotide sequences. In this aspect, thecycle numbers indicate whether MRSA is present in a sample.

In still another aspect, the invention provides a method of identifyingbacteria in a sample. This method includes the steps: (i) determiningwhether a mecA polynucleotide is present in the sample; (ii) determiningwhether an MSSA-orfX polynucleotide is present in the sample; and (iii)determining whether an S. aureus-specific polynucleotide is present inthe sample, where the S. aureus-specific polynucleotide is not an orfXpolynucleotide. In this aspect, the combination of (a), (b), and (c)present in the sample identifies bacteria in the sample.

In one aspect, the invention provides a kit for identifying MRSA in asample. Such a kit includes: a first set of primers complementary to atleast a portion of a mecA polynucleotide sequence; a second set ofprimers complementary to at least a portion of MSSA-orfX polynucleotidesequence; a third set of primers complementary to at least a portion ofan S. aureus-specific polynucleotide sequence, wherein said S.aureus-specific polynucleotide sequence is not a bridging sequence; andat least one member selected from: a DNA polymerase enzyme, dNTPs,magnesium and a stabilizer.

In another aspect, the invention provides a kit for identifying MRSA ina sample, and such a kit can include: a first set of primerscomplementary to at least a portion of a mecA polynucleotide sequence; asecond set of primers complementary to at least a portion of a bridgingsequence; a third set of primers complementary to at least a portion ofan S. aureus-specific polynucleotide sequence, wherein said S.aureus-specific polynucleotide sequence is not a bridging sequence; andat least one member selected from: a DNA polymerase enzyme, dNTPs,magnesium and a stabilizer.

In one aspect, the invention provides method of detecting anantibiotic-resistant bacterial strain in a sample. This method caninclude the steps: (i) providing a first set of primers, which arecapable of producing a first amplification product from at least aportion of a gene that confers antibiotic-resistance; (ii) providing asecond set of primers, which are capable of producing a secondamplification product from at least a portion of a bridging region;(iii) providing a third set of primers, which are capable of producing athird amplification product from at least a portion of a bacterialstrain-specific polynucleotide sequence; (iv) combining the first,second and third set of primers with said sample in a reaction mixture;(v) performing a multi-cycle amplification reaction with the reactionmixture; and (vi) determining cycle numbers of appearance of each of thefirst, second and third amplification products. In this aspect, thecycle numbers indicate whether an antibiotic-resistant bacterial strainis present in the sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a table listing sequences for primers and probes used inassays of the invention.

FIG. 2 illustrates results for detection of and discrimination betweenmethicillin resistant Staphylococcus aureus (MRSA) and methicillinsensitive Staphylococcus aureus (MSSA) using methods of the invention.

FIG. 3 illustrates results for detection of and discrimination betweencoagulase negative Staphylococcus methicillin resistant (MR-CoNS) andcoagulase negative Staphylococcus methicillin sensitive (MS-CoNS)bacteria.

FIG. 4 illustrates results from a Real Time-PCR assay of stabilized PCRreaction mixes of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention. It will be apparent to one of skill inthe art that these additional features are also encompassed by thepresent invention.

ABBREVIATIONS

“MRSA” refers to “methicillin-resistant Staphylococcus aureus”.

“MSSA” refers to “methicillin-susceptible Staphylococcus aureus”.

Scc:orfX refers to a region between the SCCmec insertion cassette andthe orfX region of the S. aureus genome. This region is also referred toas the “bridging region”.

DEFINITIONS

The term “primer” refers to an oligonucleotide, either natural orsynthetic, that is capable, upon forming a duplex with a polynucleotide(i.e. DNA) template, of acting as a point of initiation of nucleic acidsynthesis and being extended from one end along the template so that anextended double strand (duplex) is formed. Primers of the invention mayor may not be detectably labeled.

A “probe” is an oligonucleotide, either natural or synthetic, that isgenerally detectably labeled and used to identify complementary nucleicacid sequences by hybridization. Primers and probes of use in theinvention may have identical or different sequences.

A “strain” is a subset of a bacterial species differing from otherbacteria of the same species by an identifiable difference.

As used herein, the term “nucleic acid” is used interchangeably with theterm “polynucleotide”, “polynucleotide sequence” and “polynucleotidemolecule”. In addition, all of these listed terms may refer to a portionof a gene or to an entire gene, and the term “gene” is usedinterchangeably herein as the term “polynucleotide”, “polynucleotidesequence”, “polynucleotide molecule” and “nucleic acid”.

A “mecA polynucleotide sequence” refers to a polynucleotide thatcomprises a sequence encoding a portion or all of a mecA gene.

Overview

The present invention provides methods for detecting and analyzingantibiotic-resistant bacteria. Although the invention is describedherein primarily with respect to bacteria resistant to the antibioticmethicillin, it will be appreciated that the methods and compositions ofthe present invention can be used to detect bacteria resistant to a widerange of antibiotics, including in one non-limiting example, vancomycin.The flexibility of the assays and compositions encompassed by thepresent invention is due in part to the flexibility in the ability ofbacteria to acquire genes that provide antibiotic resistance. Theacquisition of such genes is often facilitated by plasmids, transposons,integrons and archetype insertion elements. Because bacteria acquireantibiotic resistance in known ways, it will be appreciated that themethods and compositions of the present invention can be adapted usingmethods known in the art to detect different types and strains ofbacteria that are resistant to various antibiotics. Information onacquisition of antibiotic resistance can be found, for example, inToleman, et al., (2006), Journal of Antimicrobial Chemotherapy 58:1-6;Handwerger et al., (1995), Antimicrobial Agents And Chemotherapy, pp.2446-2453; Launay, et al., (2006), Antimicrobial Agents AndChemotherapy, pp. 1054-1062, all of which are hereby incorporated byreference in their entirety for all purposes and in particular for theirteachings regarding the acquisition of antibiotic resistance inbacteria.

In one aspect, the invention provides a cycle threshold assay fordetecting the presence of methicillin-resistant bacteria in a sample. Ina further aspect, such an assay can also be used to determine whether adetected methicillin-resistant bacteria is Staphylococcus aureus (“S.aureus”). In addition, the present invention provides methods and assaysfor distinguishing between different strains of S. aureus, and fordetermining whether the strain present in a sample is an emerging strainnot previously identified and/or sequenced.

In one aspect, the cycle threshold assay of the invention utilizesamplification reactions directed to three different loci in a bacterialgenome. In one exemplary aspect, for detection of MRSA, these threedifferent loci are (i) the mecA gene, (ii) a bridging region between theSCCmec insertion cassette and the orfX gene, and (iii) an S.aureus-specific gene that is not orfX. In another exemplary embodiment,a region of a methicillin-susceptible S. aureus (MSSA) is amplified—thisregion is the region of the orfX gene surrounding the position at whichan insertion cassette would be inserted but does not have that insertioncassette (this region is also referred to herein as “MSSA-orfX”). Inthese exemplary embodiments, the cycle at which amplification productsfor each locus appears indicates whether MRSA is present in the sample.As used herein, the cycle at which an amplification product “appears”refers generally to the cycle at which the amplification product isdetectable—in exemplary embodiment, the product “appears” at the firstcycle number at which the amplification product has accumulated toenough of a concentration to be detected using methods known in the artand described further herein. In a further embodiment, the cycle numbercan also provide information as to the strain of MRSA that is present,and whether any other non-Staphylococcus mec-resistant bacteria arepresent in the sample.

Methods of the invention can be used to detect bacteria from samplesobtained from a variety of sources, including without limitation:clinical samples (e.g. a body fluid such as nasal fluids, whole blood,serum, plasma, cerebrospinal fluid, urine, lymph fluids, and variousexternal secretions of the respiratory, intestinal and genitourinarytracts, tears, saliva, milk as well as white blood cells, malignanttissues, amniotic fluid and chorionic villi), environmental samples,microbial cultures, microbial colonies, tissues, and cell cultures.Samples may also be obtained from food, from surfaces (such as floors,tables, and the like), and from airborne particles (including withoutlimitation pollen and dust). Samples of used in the present inventionmay be pure samples or impure samples. Samples for use in the inventionmay comprise a mixture from two or more distinct strains of bacteria.

In one embodiment, once a sample is obtained, polynucleotide sequencesmay be directly assayed from the sample. In another embodiment,polynucleotides, such as DNA, are first extracted from the sample usingmethods known in the art and then used in the assays of the invention.

Cycle Threshold Assay

The invention provides methods for detecting and analyzingantibiotic-resistant bacteria using a cycle threshold assay. In general,a cycle threshold assay of the invention utilizes a multi-cycleamplification reaction in which the cycle at which a particularamplification product appears relative to other co-amplified fragmentscan provide information on the strains and species of bacteria presentin the sample. In addition, the cycle threshold assay can provideinformation on the presence of a specific antibiotic-resistant strain,such as, but not limited to, bacterial strains resistant to methicillin.Although the cycle threshold assay of the invention is primarilydescribed herein in terms of real-time PCR, it will be appreciated thatother template-based amplification reactions can be adapted for use inaccordance with the present invention using methods known in the art anddescribed further herein.

The term “detecting and analyzing” encompasses confirming the presenceof a particular species or strain of bacteria as well as distinguishingbetween different species and strains of bacteria. The term alsoencompasses determining the concentration or relative concentrations ofdifferent species or strains of bacteria in a sample.

In one aspect, an amplification reaction is provided such thatnucleotide fragments from at least three different loci of a bacterialgenome are amplified. By determining the cycle number at which fragmentsfrom each locus appears, it is possible to differentiate between asample containing a single bacterial species (a single strain) and asample containing a mixture of different bacterial species and/orstrains. Generally, if a sample contains a mixture of different strainsor species of bacteria, and if the different strains or species have atleast a 10-times factor difference in their relative concentrations,amplification products from loci which are found in more than onespecies or strain of bacteria will appear at a cycle numbersignificantly removed (by 4 cycles or more) from the amplificationproducts from loci which are found in only a single species or strain ofbacteria. However, if only a single species or strain is present in thesample, (or if there is a mixture of strains or species which have lessthan a 10-times factor difference in their concentrations) theamplification products from all loci will appear at substantially thesame (within 3 cycles) point of a multi-cycle amplification cycle.

Cycle Threshold Assay: MRSA

In one aspect, cycle threshold assays of the invention are used todetect and analyze MRSA in a sample. As used herein, the term “MRSA”refers to any strain or sub-strain of Staphylococcus aureus bacteriawhich is resistant to the effects of the antibiotic methicillin byvirtue of acquiring a staphylococcal cassette chromosome mec (SCCmec)element containing a functional mecA gene. An exemplary MRSA DNAsequence is set forth in Genebank Accession No. NC_(—)002745.

It has been shown that methicillin-susceptible S. aureus (MSSA) strainsbecome MRSA strains by the acquisition of a Staphylococcal cassetteinsertion (SCCmec) element carrying the mecA gene. This cassette isgenerally (but not exclusively) obtained from other Staphylococci andnon-Staphylococci bacteria. The mecA gene provides the methicillinresistance characteristic of certain S. aureus strains. The mecA geneencodes a β-lactam-resistant penicillin-binding protein (PBP), whichtakes over the biosynthetic functions of the normal PBPs when the cellis exposed to β lactam antibiotics.

In one exemplary embodiment, the invention may provide methods andcompositions for the amplification of polynucleotide sequences (alsoreferred to herein as “fragments”) from: (i) the mecA gene locus, (ii) alocus for an S. aureus specific gene, and (iii) the locus for the“bridging region” between the 3′ end of the SCCmec region and the orfXgene. In this embodiment, if the sample contains an MRSA strain thatcannot be detected by any of the primers for the bridging region (i.e.,is a newly emerging strain that has not been identified and/or sequencedpreviously), then the mecA amplification product and the S. aureusspecific amplification product will appear at identical or substantiallyidentical cycle numbers (e.g., no more than 3 cycles apart in eitherdirection).

If the mecA fragment and the S. aureus specific gene fragment appear atsubstantially different cycle numbers (e.g., 4 or more cycles apart ineither direction), this indicates that the sample is a mixed bacterialsample comprising both S. aureus and another methicillin-resistantbacteria with at least a 10-times factor difference in theirconcentrations. This difference in cycle number appearance is generallya result of the mecA fragment being amplified from both populations ofbacteria, whereas the S. aureus-specific gene fragment would beamplified only from the S. aureus bacteria.

In another exemplary embodiment, a cycle threshold assay of theinvention amplifies provides methods and compositions for theamplification of fragments from: (i) the mecA gene locus, (ii) a locusfor an S. aureus specific gene, and (iii) the orfX region surroundingthe SCCmec insertion site for an MSSA bacteria that does not contain theSCCmec element (referred to herein as the “MSSA-orfX region”). In thisembodiment, if the S. aureus-specific fragment appears 4 or more cyclessooner than the MSSA orfX region fragment, this is an indication thatthe sample comprises a mixture of both MSSA and MRSA bacteria with atleast a 10-times factor difference in their concentrations. Thereasoning is that the orfX gene fragment will reflect amplified sequencefrom only MSSA bacteria, while the S. aureus-specific fragment willreflect the combined amplified sequence from MSSA and MRSA bacteria.

In another embodiment, if the S. aureus-specific fragment appears atsubstantially the same cycle (i.e., no more than 3 cycles apart ineither direction) as the MSSA-orfX gene fragment, this will indicatethat the sample does not comprise MRSA and instead contains a mixture ofMSSA bacteria and non-Staphylococcus mec-resistant bacteria. This isbecause both the MSSA-orfX amplification product and the S.aureus-specific amplification product would be derived only from theMSSA in the sample. In addition, if the concentration of mec-resistantbacteria in the sample is at least 10 times different than theconcentration of MSSA bacteria in the sample, mecA will appear at acycle number at least 4 cycles separate from the MSSA-orfX and S.aureus-specific fragments, because mecA would be amplified from aseparate population from the MSSA-orfX and S. aureus-specific fragments.

In the unusual case that MSSA and non-S. aureus methicillin resistantbacteria are present in the sample in similar concentrations, then theMSSA-orfX and S. aureus-specific fragment will both reflect amplifiedsequence derived only from the MSSA and should appear at substantiallyidentical cycle numbers (e.g. no more than 3 cycles apart) from eachother. In addition, as the mecA fragment will reflect amplified sequencederived only from the non-S. aureus methicillin-resistant bacteria insimilar concentration, it should also appear at a cycle numbersubstantially identical to the other two fragments (e.g. no more than 3cycles apart).

In accordance with the invention, cycle number analysis may be performedmanually or the analysis may be performed using instruments and softwareengineered for performing such a task. Manual and automated methods ofperforming cycle number analyses of the invention are known in the artand described further herein.

In one embodiment, all of the amplification reactions in the cyclethreshold assay are performed in the same aliquot of the sample. Inanother embodiment, one or more of the amplification reactions will beperformed in different aliquots of the sample.

The following describes in greater detail the specifics of the primersthat can be used in cycle threshold assays as well as other assaysdescribed herein for detecting antibiotic-resistant bacteria.

Primers Directed to mecA

In one aspect, primers directed to the mecA region of the Staphylococcusgenome are provided for cycle threshold assays of the invention. As usedherein, primers “directed to” a particular region or polynucleotidesequence refers generally to primers that are capable of amplifying aparticular region or polynucleotide sequence. In one embodiment, suchprimers are able to hybridize with and/or anneal to a particular genomicregion or polynucleotide sequence. These primers may be perfectlycomplementary to the particular genomic region or polynucleotidesequence, or they may have lesser complementarity but are neverthelessstill able to anneal/hybridize to the appropriate region and serve as astarting point for amplification reactions. Primers of the invention mayalso be used as probes in hybridization reactions.

In one embodiment, mecA primers of the invention can hybridize to a mecAgene as set forth in GenBank accession no. X52593. In a furtherembodiment, mecA primers of the invention have sequences as set forth inSEQ ID NOs: 10-11.

In one embodiment, primers directed to the mecA region used inaccordance with the invention have 100% sequence identity to SEQ ID NOs:10-11. In a further embodiment, the primers have about 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, and 99% sequence identity to SEQ ID NOs: 10-11.

Primers Directed to the Bridging Region Between SCCmec and orfX

In one aspect, methods of the invention utilize primers directed to thebridging region between the 3′ end of the SCCmec and the adjacentjunction region of the orfX gene.

It will be appreciated that a high degree of polymorphism exists both atthe 3′ end of the SCCmec and at the adjacent junction region the orfXgene, which accounts for the differentiation of at least 39 differentstrains of MRSA. For example, the Type III strain of MRSA has a uniquenucleotide sequence in the SCCmec, while type II has an insertion of 102nucleotides to the right terminus of SCCmec, as described withadditional detail by Huletsky et al (U.S. Pat. Appl. No. 20050019893).Sequence information pertaining to different strains of MRSA may befound in: Ito et al., (2001), Antimicrob. Agents Chemother.45:1323-1336; Hiramatsu et al., (1996), J. Infect. Chemother. 2:117-129and Ma et al., (2002), Antimicrob. Agents Chemother. 46:1147-1152 andHuletsky et al (U.S. Pat. Appl. No. 20050019893), all of which arehereby incorporated by reference in their entirety for all purposes andin particular for their teachings regarding sequences for differentstrains of MRSA.

Thus, the present invention contemplates the use of a battery of primers(also referred to herein, as bridging primers) that are capable ofdetecting the bridging region in different strains of MRSA, includingthe most promiscuous strains (types I-IV) and newly emerging strains.

Exemplary primers directed to bridging regions are set forth by SEQ IDNOs: 2-8. In one embodiment, at least 2 bridging primers are used inassays of the present invention. In a further embodiment, at least 5bridging primers are used in assays of the present invention. In stillfurther embodiments, about 3 to about 30, about 5 to about 25, about 7to about 20, about 9 to about 15 and about 10 to about 12 bridgingprimers are used in accordance with the present invention.

In one embodiment, primers directed to the bridging region have 100%sequence identity to SEQ ID NOs: 2-8. In a further embodiment, theprimers have about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99%sequence identity to SEQ ID NOs: 2-8.

Primers Directed to MSSA-orfX

As described herein, in one aspect, the invention provides assays inwhich locus is amplified using a primer set directed to the orfX regionsurrounding the SCCmec insertion site as would uniquely be found in aMSSA bacteria not containing the SCCmec element. This region is alsoreferred to herein as “MSSA-orfX”. Exemplary primers capable ofamplifying a DNA fragment comprising the MSSA-orfX are set forth by SEQID NOs: 2 and 24.

Primers Directed to an S. aureus Specific Locus

A primary weakness of traditional detection methods for rapidly evolvingbacteria, such as MRSA, is that amplification of one or two loci canresult in false negatives, particularly when a sample contains a newlyemerging strain of the target bacteria. In one exemplary aspect, thepresent invention provides methods and compositions for avoiding suchfalse negatives through amplification of three different loci, includingan S. aureus-specific locus. In one embodiment, the S. aureus-specificlocus is not within the orfX or bridging region of the bacterial genome.Examples of S. aureus-specific genes include, but are not limited to:nuc, Sa442 and femB. Exemplary primers capable of amplifying a DNAfragment comprising a nuc polynucleotide sequence are set forth by SEQID NOs: 18 and 19. Exemplary primers capable of amplifying a DNAfragment comprising a Sa442 polynucleotide sequence are set forth by SEQID NOs: 13 and 14.

In one aspect, amplification of a third S. aureus-specific locus allowsthe use of fewer primers directed to the bridging region between SCCmecand orfX (e.g. primers of SEQ ID NOs: 2-8) while still retaining theaccuracy of the cycle threshold assay in detecting not only the mostpromiscuous strains of MRSA, but also other strains of MRSA, includingnewly emerging strains. Achieving such a high degree of accuracy usingbridging primers alone could entail the use of a vast number ofadditional bridging primers, which could in turn reduce theeffectiveness and efficiency of a multiplex PCR reaction.

Primers Used as an Internal Control

It will be appreciated that assays of the present invention may alsoinclude an internal control to confirm the competence of the PCRreaction components (i.e. DNA polymerase, deoxynucleotides etc.) and toconfirm that the DNA extraction procedure does not contain any PCRinhibitors which could result in a false negative. Thus, the presentinvention provides assays which include amplification of polynucleotidesequences unrelated to the target regions of the bacterial genome. Forexample, human polynucleotide sequences, such as those related tohousekeeping genes or a well-characterized gene such as β-globin may beused as internal controls in assays of the invention. Exemplary primersthat may be used as such internal controls are set forth by SEQ ID NOs:16 and 17.

In one embodiment, amplification reactions for such internal controlloci are conducted in the same aliquot of the sample as otheramplification reactions for the cycle threshold assay of the invention.In another embodiment, the internal control amplification reaction isconducted in a different aliquot of the sample than the otheramplification reactions for the cycle threshold assay.

Cycle Threshold Assay: Vancomycin

As described above for detection and analysis of bacteria resistant tomethicillin, the present invention encompasses similar methods andcompositions for the detection and analysis of bacteria resistant toother antibiotics, including vancomycin.

As is the case for many types of antibiotic-resistance,vancomycin-resistance is conferred by insertion into the bacterialgenome of an element containing a functional van gene. Cycle thresholdassays of the invention can be used to detect and analyze bacteria thatare vancomycin resistant through detection of a region of the bacterialgenome containing at least a portion of a van gene. In one exemplaryembodiment, a cycle threshold assay of the invention will utilizeprimers capable of amplifying the van gene region. Such primers aredesigned according to methods described herein and known in the art. Inone exemplary embodiment, such primers may by complementary to a portionof the van gene. In another exemplary embodiment, such primers may becomplementary to polynucleotide sequences outside of the van gene regionbut that are nevertheless capable of amplifying the van gene region. Thevan gene region is known in the art, and vancomycin-resistance isconferred by a number of genes, including for example vanA, vanB1, andvan B2.

In a further embodiment, cycle threshold assays of the invention willutilize primers capable of amplifying van as well as two other loci inthe bacterial genome. The other two loci amplified in such an embodimentmay include bridging regions between the van region and the insertionpoint and regions of the bacterial genome that are species and/orstrain-specific. For exemplary bridging region polynucleotide sequences,see Launay et al., (2006) Antimicrob. Agents and Chemother. 50(3):1054-62, which is hereby incorporated by reference in its entirety forall purposes and in particular for its disclosure of targetpolynucleotide sequences in vancomycin resistant bacterial strains, someof which are listed in FIG. 1 as SEQ ID NOs: 25-38. Primerscomplementary to sequences such as those listed in FIG. 1 may be used incycle threshold assays and triple locus assays of the invention, asdisclosed herein. In some embodiments, such primers have 100% sequenceidentity to the sequences listed in FIG. 1 (SEQ ID NOs: 25-38). In afurther embodiment, the primers have about 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, and 99% sequence identity to SEQ ID NOs: 25-38.

Another locus that can be amplified according to methods of theinvention is a locus for a gene that is specific for a particularspecies or strain of bacteria that has acquired vancomycin resistance.For example, vancomycin resistance has been detected in strains ofStaphylococcus aureus, Oerskovia turbata, Aracanobacterium haemolyticum,Streptococcus bovis, Streptococcus gallolyticus, Streptococcuslutetiensis, Bacillus circulans, Paenibacillus, Rhodococcus,Enterococcus, as well as anaerobic bacteria belonging to the Clostridiumgenus and Eggerthella lenta. Cycle threshold assays of the invention mayinclude primers capable of amplifying genes and loci that are specificto these strains. Thus, primers directed to strain-specific loci,together with primers directed to the bridging region and a van generegion, a cycle threshold assay as described herein can be used todetect the presence of vancomycin resistant bacteria in a sample and toidentify the species and/or strain of the vancomycin-resistant bacteriain the sample.

Triple Locus Assay

A triple locus assay of the invention utilizes amplification reactionssimilar to those described herein for the cycle threshold assay. In atriple locus assay of the invention, primers directed to three differentloci are used in amplification reactions to determine which of the threeloci are present in a sample. The combination of loci detected providesinformation on whether an antibiotic-resistant bacterial strain ispresent in the sample. In a further embodiment, the combination of locidetected also provides information on whether different strains and/orspecies of bacteria are present within a sample.

In one aspect, the triple locus assay of the invention amplifies: (i) aregion comprising a gene that confers antibiotic-resistance; (ii) a“bridging region” between a gene that confers antibiotic resistance andthe point in the genome at which an element containing that gene isgenerally inserted; and (iii) a region comprising a strain-specificgene.

In one exemplary aspect, the invention provides a triple locus assaythat can be used to distinguish between MRSA and other mecA genecarrying bacteria in a sample. The triple-locus assay of the presentinvention provides an advantage over traditionally used methods ofdetecting MRSA in a sample, because the triple locus assay of theinvention has a lower possibility of producing a false-negative orfalse-positive result. In addition, the assay of the invention is ableto identify novel emerging strains of MRSA and distinguish these strainsfrom other Staphylococcus species.

In one aspect, the triple locus assay of the invention includes methodsand compositions by which three different loci of the bacterial genomeare amplified: (1) at least a portion of the mecA region; (2) at least aportion of the orfX sequence; and (3) at least a portion of an S. aureusspecific gene that is not orfX. In one embodiment, polynucleotidesequences from these loci are amplified utilizing primers such as thosedescribed herein which are directed to those regions of the bacterialgenome. In another embodiment, one or more of the amplification productsfor the three loci are detected using probes that are able to hybridizeto those regions. Such probes may have the same or different sequencesas the primers used in the amplification reactions, and can be used todetect the presence of these loci using methods well established in theart.

In one aspect, the combination of the three loci detected in a triplelocus assay of the invention provides information as to whether MRSA ispresent in the sample, whether other, non-S. aureus mecA-carryingbacteria are present in the sample, and whether MSSA is present in thesample. In a further aspect, the triple locus assay will identifywhether the strain(s) of MRSA present in a sample are novel, emergingstrains.

According to one exemplary aspect of the present invention, of the threeloci interrogated in the assay, two DNA sequences, (the first comprisinga mecA polynucleotide sequence and the second comprising the bridgingregion) are amplified. Detection of both sequences indicates that theStaphylococcus aureus bacteria present in the sample comprise aStaphylococcus aureus bacteria with SCCmec and a mecA gene and are mostlikely resistant to methicillin. Detection of only the mecA sequenceindicates that the sample comprises one or more strains ofnon-Staphylococcus aureus bacteria that carry the mecA gene and are mostlikely methicillin-resistant. Detection of only the bridging sequenceindicates that there are Staphylococcus aureus bacteria in the samplewhich do not comprise an MRSA-type SCCmec which includes the mecA gene(i.e. MSSA). Table 1 below provides a summary of some of the possibleoutcomes of a triple locus assay according to this aspect of the presentinvention:

TABLE 1 S. aureus- MecA Bridging region specific gene (fragment 1)(fragment 2) (fragment 3) Result A Yes Yes Yes Sample contains MRSA andmay also contain one or more bacteria carrying the mecA gene B Yes NoYes Sample may comprise a strain of MRSA undetectable by bridging regionprimers or sample may comprise a mixture of MSSA and a second non-S.aureus bacteria that is methicillin-resistant C No Yes or No Yes or NoSample does not contain MRSA, but may contain an atypical strain of MSSA(if both fragment 2 and fragment 3 are present) D Yes No No Sample doesnot contain MRSA but does contain methicillin- resistant non-S. aureusbacteria.

As can be seen from Table 1, in the event of outcome “A”, “C” or “D”, aclear-cut result is obtained and no further analysis is required—in theevent of outcome “A” it can be deduced that the sample comprises MRSA.In the event of outcome “C” or “D”, it can be deduced that the samplemost likely does not comprise MRSA. However, in the event of outcome B,no deduction can be made with any degree of certainty. In the case ofsuch an ambiguous outcome, a cycle threshold assay as described hereincan be used to further identify the bacteria present in the sample.

In another exemplary aspect, the invention provides methods andcompositions for amplifying the following loci: a mecA polynucleotidesequence, an S. aureus-specific polynucleotide sequence that is not theorfX gene (also referred to herein as “SA”), and an orfX polynucleotidesequence of the orfX gene surrounding the integration site of the SCCmecas would be found in a methicillin-sensitive S. aureus bacteria thatdoes not contain an SCCmec element (also referred to herein as“MSSA-orfX”). Detection of the first two sequences together withnon-detection of the third sequence indicates that the sample containsStaphylococcus aureus bacteria resistant to methicillin (i.e. MRSA).Detection of the SA specific sequence and the MSSA-orfX sequencetogether with non-detection of the mecA sequence indicates that thesample comprises Staphylococcus aureus bacteria that are notmethicillin-resistant (i.e. MSSA). Detection of mecA and the MSSA-orfXsequences and the absence of the SA sequence indicate that the samplecontains a mutated MSSA and also contains methicillin-resistant bacteriaother than S. aureus. Detection of amplification products from a singleone of the regions and the absence of amplification products from theother two regions indicates that the sample does not contain MRSA.

The amplification reactions for each locus in the triple locus assay ofthe present invention may be conducted in the same tube (i.e. using asingle aliquot of the sample) or two or more separate tubes (i.e. usinga second and or third aliquot of the sample). In one exemplaryembodiment, the amplification of all three sequences is conductedsimultaneously in the same tube (i.e. as a multiplex reaction).

Table 2 below provides a summary of some of the possible outcomes of theassay according to this aspect of the present invention:

TABLE 2 MecA SA MSSA-orfX (fragment 1) (fragment 2) (fragment 3) ResultA Yes Yes No Sample contains MRSA B No Yes Yes Sample does not containMRSA but does contain MSSA C Yes No Yes Sample does not contain MRSA butdoes contain methicillin-resistant non-S. aureus bacteria and anatypical strain of MSSA D Yes No No Sample does not contain MRSA butdoes contain methicillin-resistant non-S. aureus bacteria E No No NoSample does not contain MRSA or any Staphylococcus or othermethicillin-resistant bacteria F No Yes No Sample does not contain MRSAbut does contain an atypical strain of MSSA G No No Yes Sample does notcontain MRSA but does contain an atypical strain of MSSA H Yes Yes YesSample contains MSSA and also contains one or more bacteria carrying themecA gene, possibly MRSA, or a methicillin-resistant non-S. aureusbacteria

As can be seen from Table 2, in the event of outcome “A”, “B”, “C”, “D”,“E”, “F” or “G” a clear-cut result is obtained and no further analysisis required—in the event of outcome “A” it can be deduced that thesample comprises MRSA. In the event of outcome “B”, “C” “D”, “E”, “F” or“G”, it can be deduced that the sample most likely does not compriseMRSA. However, in the event of outcome H, a mixed sample is evident thatcontains MSSA and one or more bacterial strains carrying the mecA gene,but it is unclear if the methicillin-resistant bacteria reflect MRSA, anon-S. aureus bacteria, or both. In the case of an ambiguous result suchas outcome H, a cycle threshold assay of the invention can be used toidentify bacteria present in the sample.

In one embodiment, triple locus assays of the invention can be used todistinguish between different strains of bacteria in a sample. As shownin FIG. 2, MRSA and MSSA bacteria can be distinguished based ondetection of the products of amplification reactions directed to thebridging region, the mecA gene region, an S. aureus-specificpolynucleotide sequence (in this embodiment, the nuc gene), and a humanbeta globin polynucleotide sequence (used as an internal control). Inthe MRSA samples, all four amplification products were detected. Incontrast, in the MSSA samples, only the S. aureus-specific and theinternal control human beta globin polynucleotide were detected.

Similarly, FIG. 3 shows the results of assays which distinguish betweencoagulase negative Staphylococcus methicillin resistant (MR-CoNS) andcoagulase negative Staphylococcus methicillin sensitive (MS-CoNS)bacteria. Again, in these experiments, the amplification reactions weredirected to the bridging region, the mecA gene region, an S.aureus-specific polynucleotide sequence (in this embodiment, the nucgene), and a human beta globin polynucleotide sequence (used as aninternal control). In this case, for the samples containing MR-CoNS, themecA gene polynucleotide sequence and the internal controlpolynucleotide sequences were detected, whereas in samples containingMS-CoNS, only the internal control polynucleotide sequence was detected.Thus, the assays of the present invention provide the ability todistinguish among MRSA, MSSA, MR-CoNS and MS-CoNS bacteria.

Advantages of the Triple-Locus Assay

As discussed herein, triple locus assays of the invention are moreaccurate than traditional methods of detecting antibiotic-resistantbacteria. In particular, triple locus assays result in significantlyfewer false positive and negative results than double or single locusassays.

Double locus assays, (see e.g., Reischlet al., J. Clin. Microbiol.38:2429-2433), which are generally based on the detection of the mecAgene and S. aureus-specific chromosomal sequences often encounterdifficulty in discriminating MRSA from methicillin-resistantcoagulase-negative staphylococci (MR-CoNS), because the mecA gene iswidely distributed in both S. aureus and CoNS species. As more than 90%of nasal swabs contain staphylococci, (usually coagulase-negativestaphylococci), and as 70-80% of CoNS are MR-CoNS (Becker et al, (2006),J. Clin. Microbiol. 44:229-231; Diekema et al, (2001), Clin. Infect.Dis. 32(Suppl. 2):S114-S132), many mixed population samples containingMSSA and MR-CoNS may be mistakenly identified as MRSA. Consequently,double locus assays are characterized by a high percentage of falsepositive MRSA with a low positive predictive value (PPV), and usuallycannot be applied on specimens potentially containing a mixed bacterialpopulation.

Single-locus assays have been suggested to overcome the above-describeddifficulties in the double locus assays. Single locus assays aregenerally based on the detection of the right extremity of the SCCmecelement inserted adjacent to the S. aureus-specific orfX gene (see e.g.,Huletsky et al, (2004), J. Clin. Microbiol. 42:1875-1884). However, newstrains of MRSA are constantly emerging with mutations on both sides ofthis junction (Hansse et al., (2004), Antimicrobial Agents Chemotherapy,48:285-96; Sundsfjord et al., (2004), APMIS, 112:815-8137; Witte et al.,(2005), Eur J Microbiology and Infectious Diseases., 24:1-5). As such,single locus assays for MRSA are characterized by false negatives.Furthermore, the single-locus assay cannot differentiate between aSCCmec element comprising the mecA gene and an SCCmec which does notcarry the mecA gene.

Hiramatsu et al (U.S. Pat. No. 6,156,507) describe a single locus assaydesigned to detect amplification of the orfX gene surrounding theintegration site of the SCCmec in the absence of SCCmec insertion. Inthe Hiramatsu et al. assay, a negative finding is interpreted asindicative of the presence of MRSA. An assay based on a negative resultis an inadequate method of determining the presence of MRSA, as anegative finding could also be obtained from a patient sample containingneither MSSA nor MRSA. Furthermore, as Hiramatsu et al point out, apatient's sample containing a mixture of MRSA and MSSA would give apositive result in these tests and be falsely interpreted as being freeof MRSA. In addition, studies have shown that Hiramatsu's single locusassay lacks in clinical adequacy due to its failure to correctly detectMRSA from clinical samples reflecting MRSA strains containing SCCmectypes other than I-V (see e.g., Hansse et al., (2004), AntimicrobialAgents Chemotherapy, 48:285-96; Sundsfjord et al., (2005) APMIS,112:815-8137, 2004; Witte et al., (2005) Eur J Microbiology andInfectious Diseases, 24:1-5).

Another weakness of a single-locus assay is that such an assay cannotdifferentiate between bacteria comprising an SCCmec element whichcarries the mecA gene (MRSA) or an SCC element that does not carry themecA gene (such as SCCcap present in several MSSA strains), which canthus result in a false positive. Therefore, a single locus assay ofsamples comprising an MSSA which carries an SCC element that does notcomprise a mecA gene can result in a false positive. The potential forfalse positive findings undermine the clinical utility of thesingle-locus assay as a reliable method of screening patients as part ofan infection control program. Indeed, numerous studies utilizing thesingle-locus assay report clinical examples of MSSA presenting falsepositive as pseudo-MRSA (Rupp, et al., J Clinical Microbiology, 2006,44:2317; Deplano et al., J Antimicrobial Chemotherapy 2000, 46: 617-620;Corkill, et al. J Antimicrobial Chemotherapy 2004, 54:229-231, Lina etal. 1999, Clini. Infect. Dis. 29:1128-1132; Warren et al. J. Clin.Microbiol. 42:5578-5581).

Methods of the present invention, including both the triple-locus assayand the cycle threshold assay, show an increased accuracy overtraditional methods of detecting antibiotic-resistant bacteria such asMRSA. As illustrated in Example 3, the triple locus assay of the presentinvention was shown to be 99.6% sensitive and 97.4% specific, displayinga degree of accuracy far greater than either the single or double locusassays described hereinabove and further in Examples 1 and 2.

Methods of Amplification and Detection

Both the cycle threshold and triple locus assays of the inventioninclude amplification reactions that utilize primers capable ofamplifying selected regions of a genome. The term “capable ofamplifying” describes primers that are able to produce an amplificationproduct that includes a selected region of the bacterial genome. Primerscapable of amplifying a selected region include primers that arecomplementary to a sequence within the target region. Primer capable ofamplifying a selected region also include primers that are complementaryto regions that overlap at least a portion of the target region, as wellas primers that are complementary to regions outside of a target regionbut that are nevertheless able to produce an amplification product thatincludes the target region. It will be appreciated that such primers canbe designed using methods well-established in the art.

Amplification methods used in the assays of the present inventionencompass any method capable of amplifying a targeted portion of apolynucleotide sequence. Such amplification methods include withoutlimitation: polymerase chain reaction (PCR), nucleic acid sequence-basedamplification (NASBA), self-sustained sequence replication (3SR), stranddisplacement amplification (SDA) and branched DNA signal amplification(bDNA).

In one embodiment, PCR is used as the amplification method in assays ofthe invention. PCR is an in vitro technique for the enzymatic synthesisof specific DNA sequences using two oligonucleotide primers thathybridize to complementary nucleic acid strands and flank a region thatis to be amplified in a target DNA. A series of reaction steps,including (1) template denaturation, (2) primer annealing, and (3)extension of annealed primers by DNA polymerase, results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers.

The term “PCR” as used herein encompasses derivative forms of thereaction, including but not limited to real-time PCR, quantitative PCR,multiplexed PCR, reverse transcription PCR and the like. Reactionvolumes can range from about 100 nanoliters (nl) to about 500microliters (μl), from about 200 nl to about 250 μl, from about 500 nlto about 200 μl, from about 1 μl to about 100 μl, from about 10 μl toabout 80 μl, from about 20 μl to about 60 μl, from about 5 to about 30μl, from about 10 to about 25 μl, and from about 30 μl to about 40 μl.

“Real-time PCR” refers to a PCR method in which the amount of reactionproduct, i.e. amplicon, is monitored as the reaction proceeds. There aremany forms of real-time PCR, which differ mainly in the detectionchemistries used for monitoring the reaction product, e.g. Gelfand etal, U.S. Pat. No. 5,210,015 (“TaqMan®”); Wittwer et al, U.S. Pat. Nos.6,174,670 and 6,569,627 (intercalating dyes, such as SYBER® Green);Tyagi et al, U.S. Pat. No. 5,925,517 (molecular beacons); all of whichare incorporated herein by reference in their entirety for all purposesand in particular for their teachings regarding real-time PCR. Otherexemplary detection chemistries include, but are not limited to ScorpionPrimers, Sunrise Primers, and Eclipse Probes. Detection chemistries forreal-time PCR are reviewed in Mackay et al, (2002) Nucleic AcidsResearch, 30:1292-1305, which is also incorporated herein by referencein its entirety for all purposes, and in particular for its disclosureof different detection chemistries for real-time PCR.

“Multiplexed PCR” refers to a PCR wherein multiple sequences aresimultaneously amplified in the same reaction mixture (multi-colorreal-time PCR). Generally in such methods, distinct sets of primers areemployed for each sequence being amplified.

“Quantitative PCR” refers to a PCR designed to measure the abundance ofone or more specific target sequences in a sample or specimen.Quantitative PCR includes both absolute quantitation and relativequantitation of such target sequences. Quantitative measurements aremade using one or more reference sequences that may be assayedseparately or together with a target sequence. The reference sequencemay be endogenous or exogenous to a sample or specimen, and in thelatter case, may comprise one or more competitor templates. Typicalendogenous reference sequences include segments of transcripts of thefollowing genes: β-actin, GAPDH, β₂ microglobulin, ribosomal RNA, andthe like. Techniques for quantitative PCR are well-known to those ofordinary skill in the art, as exemplified in the following referencesthat are incorporated by reference in their entirety: Freeman et al,(1999) Biotechniques, 26: 112-126; Becker-Andre et al, (1989) NucleicAcids Research, 17: 9437-9447; Zimmerman et al., (1996) Biotechniques,21: 268-279; Diviacco et al, (1992) Gene, 122: 3013-3020.

Generally amplification methods used in methods of the invention willutilize primers as starting points for the amplification of the templatein each cycle of the reaction. In such reactions, primers anneal to acomplementary site on the template (also referred to herein as “target”)polynucleotide and then enzymes such as DNA polymerase are used toextend the primers along the sequence of the template polynucleotide.Primers typically have a length in the range of from about 5 to about50, about 10 to about 40, about 12 to about 30, and about 20 to about 25nucleotides. The length of the primers is typically selected such thatthe primers bind with optimal selectivity to a target polynucleotidesequence.

Generally, primers are used as pairs which include a ‘forward’ primerand a ‘reverse’ primer, with the amplification target of interest lyingbetween the regions of the template polynucleotide that arecomplementary to those primers. The design and selection of appropriatePCR primer sets is a process that is well known to a person skilled inthe art. Automated methods for selection of specific pairs of primersare also well known in the art, see e.g. U.S. Publication No.20030068625. In one embodiment, a set of amplification primers can beselected such that the distance between the two primers (i.e. the lengthof the amplicon) is at least 5 base pairs. In another embodiment, theprimers are selected such that the distance is about 5 to about 50,about 10 to about 40, and about 20 to about 30 base pairs. In oneembodiment, amplicons resulting from real-time PCR methods are fromabout 50 to about 400 bp, from about 75 to about 300, from about 100 toabout 200 and from about 180 to about 400 bp.

In one aspect, the products of amplification reactions are detectedusing labeled primers. In another aspect, such products are detectedusing probes directed to particular regions of the template nucleicacid. In still another aspect of the present invention, the assay is amolecular-beacon based assay. Molecular beacons are hairpin-shapedoligonucleotide probes that report the presence of specific nucleicacids in homogeneous solutions. When they bind to their targets theyundergo a conformational reorganization that restores the fluorescenceof an internally quenched fluorophore (Tyagi et al., (1998) NatureBiotechnology. 16:49]. Exemplary probe sequences that may be used inassays of the present invention are set forth by SEQ ID NOs: 1, 9, 12,15 and 20.

Kits

In one aspect, the invention provides kits that include components forperforming the assays described herein.

In one embodiment, the invention provides kits comprising reaction mixesfor use in real time amplification assays. Such reaction mixes can bestabilized mixes containing all the constituents for performing thereaction in one or more containers (such as tubes for use in a PCRmachine). In an exemplary embodiment, such stabilized reaction mixesinclude primers, fluorescently labeled probes. In a further embodiment,such mixes are stabilized such that they can be stored at roomtemperature.

It will be appreciated that kits of the present invention may includeprimers for use in assays of the invention to detect and analyzeantibiotic-resistant bacteria. In one embodiment, kits of the inventioninclude primers of use for the identification of MRSA. A kit of thepresent invention may, if desired, be presented in a pack which maycontain one or more units of the kit of the present invention. The packmay be accompanied by instructions for using the kit. The pack may alsobe accommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of laboratory supplements, which notice is reflective of approvalby the agency of the form of the compositions.

A kit of the present invention may comprise primers for amplifying apolynucleotide sequence from mecA and bridging regions, as describedherein. The kit may also comprise primers for amplifying apolynucleotide sequence for an S. aureus-specific gene and primers foramplifying a region used as an internal control, which could encode, forexample, a human gene. The kit may also comprise other components of thePCR reaction including but not limited to DNA polymerase, dNTPs,magnesium, buffers and stabilizers.

According to one embodiment, a kit of the invention will include amixture containing the components of a PCR reaction as described above.In a further embodiment, such a mixture is dehydrated by at least 50%.The dehydrating may be effected by any means known in the art,including, but not limited to: oven heating, lyophilization, and vacuumhydration removal. According to a still further embodiment, at least oneof the amplification primers and probes of the present invention areincorporated into such a PCR reaction mixture prior to dehydration.

According to yet another embodiment, the reaction components includingDNA polymerase, dNTPs, magnesium and stabilizers and all the reactionprimers and probes to be used in an assay of the present invention maybe dehydrated by at least 50% to form a ready-to-use mix that can bestored at room temperature without degradation for at least 40 days.Such a mix may be stored in a microtube, a microtube strip, or amulti-well plate. Kits comprising such dehydrated components can beutilized by laboratory personnel with limited training and experiencewith reduced risk of carry-over cross contamination or experimentalerror. Furthermore, the kits of the present invention can be transportedwithout a need for packing in dry-ice, enabling easier delivery.

An example of the shelf-life performance of a stabilized reactionmixture in kits of the invention is shown in FIG. 4. In theseexperiments, reaction mixtures stored at room temperature were assayedafter 5 and 10 weeks of storage and were compared to samples stored at−20° C. FIG. 4A shows the results from RNase P gene amplification and 4Bshows the results from human beta globin gene amplification. For bothamplification reactions, the stabilized (room temperature storage) mixesshowed similar results to the reaction mixes stored at −20° C. at boththe 5 week and 10 week time points.

Embodiments and Combinations Encompassed in the Scope of the Invention

In one aspect, the invention provides a method of detectingmethicillin-resistant S. aureus (MRSA) in a sample. In this aspect, themethod includes the steps: (i) providing a first set of primers wherethe primers are complementary to at least a portion of a mecApolynucleotide sequence; (ii) providing a second set of primers, wherethe primers are complementary to at least a portion of a bridgingregion; (iii) providing a third set of primers, where the primers arecomplementary to at least a portion of an S. aureus-specificpolynucleotide sequence, and where the S. aureus-specific polynucleotidesequence is not an orfX polynucleotide; (iv) combining the first, secondand third set of primers with the sample in a reaction mixture; (v)performing a multi-cycle amplification reaction with the reactionmixture; and (vi) determining cycle numbers of appearance of each of themecA, bridging region and S. aureus-specific polynucleotide sequences.In this aspect, the cycle numbers indicate whether MRSA is present in asample.

In accordance with the above, the invention further provides a method inwhich the first set of primers includes a plurality of primers withsequences according to at least one of SEQ ID NOs: 10-11.

In accordance with any of the above, the invention further provides amethod in which the second set of primers includes a plurality ofprimers with sequences according to at least one of SEQ ID NOs: 2-8.

In accordance with any of the above, the invention further provides amethod in which the third set of primers includes a plurality of primerswith sequences according to at least one of SEQ ID NOs: 13-14 and 18-19.

In accordance with any of the above, the invention further provides amethod in which the amplification reaction is a real-time polymerasechain reaction (PCR).

In accordance with any of the above, the invention further provides amethod which includes the additional steps: contacting the sample withprimers complementary to at least a portion of a human genepolynucleotide sequence; amplifying the human gene polynucleotidesequence; and detecting the amplified human gene polynucleotidesequence.

In accordance with any of the above, the invention further provides amethod in which the S. aureus-specific polynucleotide sequence is atleast a portion of a gene, which is a member selected from nuc, Sa442,and femB.

In accordance with any of the above, the invention further provides amethod in which the S. aureus-specific polynucleotide sequence is atleast a portion of the gene nuc.

In accordance with any of the above, the invention further provides amethod in which the S. aureus-specific polynucleotide sequence is atleast a portion of the gene Sa442.

In another aspect, the invention provides a method of detectingmethicillin-resistant S. aureus (MRSA) in a sample. In this aspect, themethod includes the steps: (i) determining whether a mecA polynucleotideis present in the sample; (ii) determining whether a bridging regionpolynucleotide is present in the sample; and (iii) determining whetheran S. aureus-specific polynucleotide is present in the sample. In thisaspect, the S. aureus-specific polynucleotide is not an orfXpolynucleotide. In this aspect, if the mecA polynucleotide, the bridgingregion polynucleotide, and the S. aureus-specific polynucleotide are allpresent in the sample, then MRSA is present in the sample.

In accordance with the above, the invention further provides a method inwhich determining whether the mecA polynucleotide is present in thesample includes conducting an amplification reaction on the sample. Thisamplification reaction utilizes a first set of primers, and a majorityof the first set of primers are complementary to at least a portion ofthe mecA polynucleotide.

In accordance with any of the above, the first set of primers includesat least one of SEQ ID NOs. 10-11.

In accordance with any of the above, the invention further provides amethod in which determining whether the orfX polynucleotide is presentin the sample includes conducting an amplification reaction on thesample, wherein the amplification reaction utilizes a second set ofprimers, and wherein a majority of the second set of primers arecomplementary to at least a portion of the orfX polynucleotide.

In accordance with any of the above, the invention further provides amethod in which the second set of primers includes at least one of SEQID NOs. 2-8.

In accordance with any of the above, the invention further provides amethod in which determining whether the S. aureus-specificpolynucleotide is present in the sample includes conducting anamplification reaction on the sample, wherein the amplification reactionutilizes a third set of primers, and wherein a majority of the third setof primers are complementary to at least a portion of the S.aureus-specific polynucleotide.

In accordance with any of the above, the invention further provides amethod in which the third set of primers includes at least one of SEQ IDNOs. 13-14 and 18-19.

In accordance with any of the above, the invention further provides amethod in which the S. aureus-specific polynucleotide is at least aportion of a gene, which is a member selected from nuc, Sa442, and femB.

In accordance with any of the above, the invention further provides amethod in which the S. aureus-specific polynucleotide is nuc.

In accordance with any of the above, the invention further provides amethod in which the S. aureus-specific polynucleotide is Sa442.

In accordance with any of the above, the invention further provides amethod which includes the additional steps of: contacting the samplewith primers complementary to at least a portion of a human genepolynucleotide sequence; amplifying the human gene polynucleotidesequence; and detecting the amplified human gene polynucleotidesequence.

In accordance with any of the above, the invention further provides amethod in which the human gene polynucleotide sequence includes at leasta portion of a housekeeping gene.

In accordance with any of the above, the invention further provides amethod in which the human gene polynucleotide sequence includes at leasta portion of a β-globin gene.

In accordance with any of the above, the invention further provides amethod in which all three polynucleotide sequences are detectedsimultaneously.

In accordance with any of the above, the invention further provides amethod in which determining whether each of the three polynucleotidesequences are present in a sample is accomplished sequentially in anyorder.

In accordance with any of the above, the invention further provides amethod in which determining whether each of the three polynucleotidesequences are present in a sample is accomplished in an identicalaliquot of the sample.

In accordance with any of the above, the invention further provides amethod in which determining whether each of the three polynucleotidesequences are present in a sample is accomplished in different aliquotsof the sample.

In accordance with any of the above, the invention further provides amethod in which if the mecA polynucleotide is present in the sample andthe S. aureus-specific polynucleotide is present in the sample but thebridging region polynucleotide is not present in the sample, then thesample includes non-Staphylococcus methicillin-resistant bacteria.

In still another aspect and in accordance with any of the above, theinvention provides a method of detecting methicillin-resistant S. aureus(MRSA) in a sample. In this aspect, the method includes the steps: (i)providing a first set of primers, where the first set of primers arecomplementary to at least a portion of a mecA polynucleotide sequence;(ii) providing a second set of primers, where the second set of primersare complementary to at least a portion of an MSSA-orfX polynucleotidesequence; (iii) providing a third set of primers, where the third set ofprimers are complementary to at least a portion of an S. aureus-specificpolynucleotide sequence, and where the S. aureus-specific polynucleotidesequence is not an orfX polynucleotide; (iv) combining the first, secondand third set of primers with the sample in a reaction mixture; (v)performing a multi-cycle amplification reaction with the reactionmixture; and (vi) determining cycle numbers of appearance of each of themecA, MSSA-orfX and S. aureus-specific polynucleotide sequences. In thisaspect, the cycle numbers indicate whether MRSA is present in a sample.

In accordance with the any of above, the invention further provides amethod in which if all three sets of primers produce amplificationproducts in an amplification reaction, and the S. aureus-specificpolynucleotide sequence appears at a cycle at least 4 cycles removedfrom appearance of the MSSA-orfX polynucleotide sequence, then thesample includes both MRSA and methicillin-sensitive S. aureus (MSSA).

In accordance with any of the above, the invention further provides amethod in which if all three sets of primers produce amplificationproducts in an amplification reaction, and the S. aureus-specificpolynucleotide sequence appears within 3 cycles of the MSSA-orfXpolynucleotide sequence, then the sample includes MSSA and a non-S.aureus methicillin resistant bacteria.

In still another aspect and in accordance with any of the above, theinvention provides a method of identifying bacteria in a sample. Thismethod includes the steps: (i) determining whether a mecA polynucleotideis present in the sample; (ii) determining whether an MSSA-orfXpolynucleotide is present in the sample; and (iii) determining whetheran S. aureus-specific polynucleotide is present in the sample, where theS. aureus-specific polynucleotide is not an orfX polynucleotide. In thisaspect, the combination of (a), (b), and (c) present in the sampleidentifies bacteria in the sample.

In accordance with any of the above, the invention further provides amethod in which if the mecA polynucleotide and the S. aureus-specificpolynucleotide are both present in the sample and the MSSA-orfXpolynucleotide is not present in the sample, then the sample includesmethicillin-resistant S. aureus (MRSA).

In accordance with any of the above, the invention further provides amethod in which if the S. aureus-specific polynucleotide and theMSSA-orfX are both present in the sample and the mecA polynucleotide isnot present in the sample, then the sample includesmethicillin-susceptible S. aureus (MSSA) but does not comprise MRSA.

In accordance with any of the above, the invention further provides amethod in which if the mecA polynucleotide and the MSSA-orfXpolynucleotide are both present in the sample and the S. aureus-specificpolynucleotide is not present in the sample, then the sample includes anon-S. aureus methicillin-resistant bacteria and the sample does notcomprise MRSA.

In accordance with any of the above, the invention further provides amethod in which if only one of the mecA polynucleotide, the MSSA-orfXpolynucleotide and the S. aureus-specific polynucleotide are present inthe sample, then the sample does not comprise MRSA.

In accordance with any of the above, the invention further provides amethod in which the sample may comprise no bacteria, bacteria from asingle strain, or a mixture of bacteria from more than one strain.

In accordance with any of the above, the invention further provides amethod in which the sample is a member selected from a bodily fluid, anasal swab, and a tissue.

In accordance with any of the above, the invention further provides amethod in which the determining whether a mecA polynucleotide is presentin the sample, the determining whether an MSSA-orfX polynucleotide ispresent in the sample, and the determining whether an S. aureus-specificpolynucleotide is present in the sample are all amplification reactionsconducted in separate aliquots of the sample.

In accordance with any of the above, the invention further provides amethod in which two or more of the determining whether a mecApolynucleotide is present in the sample, the determining whether anMSSA-orfX polynucleotide is present in the sample, and the determiningwhether an S. aureus-specific polynucleotide is present in the sampleare amplification reactions conducted in identical aliquots of thesample.

In another aspect and in accordance with any of the above, the inventionfurther provides a kit for identifying MRSA in a sample, and such a kitcan include: a first set of primers complementary to at least a portionof a mecA polynucleotide sequence; a second set of primers complementaryto at least a portion of a bridging sequence; a third set of primerscomplementary to at least a portion of an S. aureus-specificpolynucleotide sequence, wherein the S. aureus-specific polynucleotidesequence is not a bridging sequence; and at least one member selectedfrom: a DNA polymerase enzyme, dNTPs, magnesium and a stabilizer.

In another aspect and in accordance with any of the above, the inventionfurther provides a kit for identifying MRSA in a sample. Such a kitincludes: a first set of primers complementary to at least a portion ofa mecA polynucleotide sequence; a second set of primers complementary toat least a portion of MSSA-orfX polynucleotide sequence; a third set ofprimers complementary to at least a portion of an S. aureus-specificpolynucleotide sequence, wherein the S. aureus-specific polynucleotidesequence is not a bridging sequence; and at least one member selectedfrom: a DNA polymerase enzyme, dNTPs, magnesium and a stabilizer.

In one aspect and in accordance with any of the above, the inventionfurther provides method of detecting an antibiotic-resistant bacterialstrain in a sample. This method can include the steps: (i) providing afirst set of primers, which are capable of producing a firstamplification product from at least a portion of a gene that confersantibiotic-resistance; (ii) providing a second set of primers, which arecapable of producing a second amplification product from at least aportion of a bridging region; (iii) providing a third set of primers,which are capable of producing a third amplification product from atleast a portion of a bacterial strain-specific polynucleotide sequence;(iv) combining the first, second and third set of primers with thesample in a reaction mixture; (v) performing a multi-cycle amplificationreaction with the reaction mixture; and (vi) determining cycle numbersof appearance of each of the first, second and third amplificationproducts. In this aspect, the cycle numbers indicate whether anantibiotic-resistant bacterial strain is present in the sample.

In accordance with any of the above, the invention further provides amethod in which the gene amplified using a first set of primers confersresistance to vancomycin.

In accordance with any of the above, the invention further provides amethod in which the gene amplified using a first set of primers confersresistance to methicillin.

In accordance with any of the above, the invention further provides amethod in which the bacterial strain-specific polynucleotide sequenceamplified is an S. aureus-specific polynucleotide sequence.

A In accordance with any of the above, the invention further provides amethod in which the bridging region amplified includes a polynucleotidesequence that is a member selected from SEQ ID NOs: 2-8.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate preferred embodiments of the invention, but should in no waybe construed as limiting the broad scope of the invention.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in thisapplication are incorporated by reference in the entirety.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Oligonucleotide Synthesis” Gait, M. J., ed. (1984);“Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds.(1985); “Transcription and Translation” Hames, B. D., and Higgins S. J.,eds. (1984); “A Practical Guide to Molecular Cloning” Perbal, B., (1984)and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols:A Guide To Methods And Applications”, Academic Press, San Diego, Calif.(1990), all of which are incorporated by reference as if fully set forthherein. Other general references are provided throughout this document.The procedures therein are believed to be well known in the art and areprovided for the convenience of the reader. All the informationcontained therein is incorporated herein by reference.

Example 1 Single and Double Locus Assays Using Pure Culture Samples

A single locus qRT-PCR assay for detecting MRSA was performed usingmethods based on Huletsky et al (US 20050019893 and US20060252078). Thisassay uses multiple primers and a dual labeled probe for hybridizationof the right extremity junction of SCCmec:orfX. Detection of human βglobin was added as an internal control. A dual labeled probecomplementary to the S. aureus orfX region (SEQ ID NO: 1) was used. Aforward primer complementary to the S. aureus orfX region (SEQ ID NO: 2)and six reverse primers complementary to MREJ sequences in the Sccmecelement. “MREJ” refers to “poly-Morphic Right Extremity Junction”. InHuletsky et al., US Patent Publication No. 20050019893, MREJ is asequence toward the right end of the SCC mec cassette that is prone tomutation—many different MRSA strains will show variations in this regionof their genome. Types i, ii, iii, iv and v of the SCCmec gene (SEQ IDNO: 3-SEQ ID NO: 8) were used. In place of the primers taught byHuletsky et al (2004) for hybridization to MREJ Types iv (meciv511), twoslightly different primers (SEQ ID NO: 7 and SEQ ID NO: 8), eachoverlapping the sequence of meciv511 were used. This change providedcomparable detection of MREJ Type IV, but reduced non-specificbackground amplification. An internal control assay was added consistingof one probe (SEQ ID NO: 15), a forward primer (SEQ ID NO: 16) and areverse primer (SEQ ID NO: 17) in order to detect human β globin.

For the double locus assay, a qRT-PCR assay for detecting MRSA wasperformed as described by Reischl et al (2000), J. Clin. Microbiol.38:2429-2433. This assay uses primers and probes to detect the S. aureusspecific gene Sa442 and the mecA gene. Detection of human β globin wasadded as an internal control. A dual labeled probe was used with asequence as set forth in SEQ ID NO: 12 for the detection of the S.aureus specific gene Sa442. A dual labeled probe was used with asequence as set forth in SEQ ID NO: 9 for the detection of mecA. Aforward primer and a reverse primer complementary to the S. aureusspecific gene Sa442 (SEQ ID NOs: 13-14) was used. A forward primer and areverse primer complementary to the mecA gene (SEQ ID NOs: 10-11) wasused. An internal control assay was added consisting of one probe (SEQID NO: 15), a forward primer (SEQ ID NO: 16) and a reverse primer (SEQID NO: 17) in order to detect human β globin.

For both the single and double locus assays, primers as described abovewere added to a standard qRT-PCR reaction mixture (Absolute™ QPCR Mixfrom ABgene) and tested with DNA samples of known MRSA, MSSA, MS-CoNSand MR-CoNS strains. Each assay was tested on 150 pure culture samples;50 MRSA, 50 MSSA, 25 MR-CoNS and 25 MS-CoNS. Amplification and detectionreactions were run using a RotorGene 3000 System Real Time PCRinstrument from Corbett Life Science according to the following standardqRT-PCR protocol:

1. 15 minute at 95° C. for enzyme activation and DNA denaturation.

2. 50 amplification cycles each consisting of the following three steps:

-   -   Step 1—10 seconds at 95° C.    -   Step 2—30 seconds at 56° C.    -   Step 3—15 seconds at 72° C. (at the end of step three the        readings were taken for each of the four fluorescent dyes)

The single locus assay yielded 51 SCC:orfX-positive results, of which 49were MRSA (true positive) and 2 were MSSA (false positive). The singlelocus assay also yielded 99 SCC:orfX-negative results, of which one wasMRSA (false negative), 48 where MSSA (true negative), 25 where MR-CoNS(true negative) and 25 where MS-CoNS (true negative). The single locusassay resulted in a sensitivity of 98%, a specificity of 98%, a positivepredictive value of 96.1% and a negative predictive value of 99% indetecting a pure culture sample.

The double locus assay yielded 50 Sa442-positive mecA-positive results,of which all 50 were MRSA (true positive). The double locus assay alsoyielded 48 Sa442-positive mecA-negative results, all 48 were MSSA (truenegative). This assay also yielded 25 Sa442-negative mecA+ results, all25 were MR-CoNS (true negative for MRSA). Double Locus Assay alsoyielded 27 Sa442-negative mecA-negative results, 25 of which wereMS-CoNS (true negative) and 2 of which were MSSA (true negative for MRSAyet it is a false negative for MSSA which is suppose to contain theSa442 gene). The double locus assay resulted in a sensitivity of 100%, aspecificity of 100%, a positive predictive value of 100% and a negativepredictive value of 100% in detecting a pure culture sample.

The above results demonstrate that primers used in accordance with thepresent invention can be successfully used to amplify target regions ofthe genomes for different species and strains of Staphylococcus.

Example 2 Demonstration of the Limited Accuracy of Single and DoubleLocus Assays in Differentiating MRSA from MSSA, MS-CoNS, and MR-CoNSUsing Clinical Patient Samples

qRT-PCR reactions were performed using the Single Locus Assay and DoubleLocus Assay as described for Example 1 herein above with 460 samples ofpatient DNA derived from cultured swabs which contain mixed populations.The reactions were run as described herein above utilizing a standardqRT-PCR reaction mixture (Absolute™ QPCR Mix from ABgene) and standardqRT-PCR protocol described above, with amplification and detection runusing a RotorGene 3000 System Real Time PCR instrument from Corbett LifeScience.

All runs contained 2 NTC samples (No Template Control), and known MRSA,MSSA, and MR-CoNS controls. Clinical samples were plated and qRT-PCR wasperformed the next day on S. aureus suspected colonies only. All sampleswere analyzed for mixed population using the same bacteriological needleused to sample colonies for qRT-PCR. All samples were analyzed also byconventional methods (plating on CNA, Mannitol and Chromeagar-MRSAplates (Hy-labs, Israel), visual inspection, slide agglutination(Pastorex, Bio-Rad) and plating on MH+NaCl+OXA and DNase plates(Hy-labs, Israel) for conformation.

338 samples were analyzed (after exclusion of patient duplicates (122)),219 of which contained MRSA, of which 37% also contained otherGram-positive bacteria, 6% Gram-negative bacteria and 32% bothGram-positive and Gram-negative. Of the 119 non-MRSA samples, 8 wereMS-CoNS, 16 MR-CoNS, 5 non-staphylococcal, and 90 MSSA's. Of the 90MSSA's 42% contained also other Gram-positive bacteria, 2% containedGram-negative bacteria and 21% contained both Gram-positive andGram-negative. Of the MSSA containing samples, 49% also containedMR-CoNS. It should be noted that these percentages do not represent realpercentage of nasal swabs mixed populations, but represent the mixedpopulation sampled for qRT-PCR, biased towards Oxacyllin resistantbacteria, as many samples originated from Oxacyllin containing plates.It is expected that the proportion of such mixtures to be much higher indirect swab analysis.

All NTC's and controls were detected correctly at all runs of bothassays.

The single locus assay displayed unsatisfactory accuracy with the mixedcultures. The single locus assay yielded 216 SCC:orfX-positive results,of which 203 were MRSA (true positive) and 13 where MSSA (falsepositive). The single locus assay also yielded 122 SCC:orfX-negativeresults, of which 16 were MRSA (false negative), 77 were MSSA (truenegative), 16 were MR-CoNS (true negative), 8 were MS-CoNS (truenegative) and 5 were non-Staphylococcus (true negative). The singlelocus assay resulted in a specificity of only 89.1% and a negativepredictive value of 86.9% in detecting a mixed culture sample. Theresults are summarized in Table 3 herein below.

TABLE 3 Convention Single-locus Test^(a) Double-locus Test^(b) CultureSCC:orfX+ SCC:orfX− Sa442+mec+ Sa442+mec− Sa442−mec+ Sa442−mec−Identification (MRSA) (other) (MRSA) (MSSA) (non S.A OXAr) (other) MRSA(n = 203 16 218 1 — — 219) MSSA (n =  13 77 (44 + 2)^(c) 35 — 9 90)MR-CoNS — 16 — — 16 (n = 16) MS-CoNS — 8 — — — 8 (n = 8) Non-Staph. — 55 (n = 5) ^(a)sensitivity = 92.7%, specificity = 89.1%, Positivepredictive value = 93.1%, Negative predicted value = 86.9%,^(b)sensitivity = 99.5%, specificity = 61.4%, Positive predictive value= 82.6%, Negative predicted value = 98.6%, ^(c)44 were MSSA mixed withMR-CoNS, 2 were MRSA with non-functional mecA genes - genetically theselast two were MRSA genetically, but phenotypically MSSA (also referredto herein as “mec-positive MSSA).

As summarized above in Table 2, the double locus assay also displayedunsatisfactory accuracy with the mixed cultures. The double locus assayyielded 264 Sa442-positive mecA-positive results, of which 218 were MRSA(true positive) and 46 were MSSA (false positive). Of the falsenegatives, 44 were determined by culture examination to be MSSA mixedwith MR-CoNS and two were found to be mecA-positive MSSA, which probablyoriginated from MRSA with mecA genes mutated to make themnon-functional. Such bacteria are genetically MRSA but phenotypicallyMSSA. The double locus assay also yielded 36 Sa442-positivemecA-negative results, one was MRSA (false negative) and 35 were MSSA(true negative). The double locus assay also yielded 16 Sa442-negativemecA-positive results, and all 16 were MR-CoNS (true negative for MRSA).The double locus assay also yielded 22 Sa442-negative mecA-negativeresults, 9 of which were MSSA (true negative), 8 of which were MSCoNS(true negative) and 5 where non-Staphylococcus (true negative). Thedouble locus assay resulted in a specificity of only 61.4% and apositive predictive value of 82.6% in detecting a mixed culture sample.

Example 3 Demonstration of Superior Accuracy of Triple Locus and CycleThreshold Assays

A triple locus high density multiplex qRT-PCR assay mixture wasdeveloped containing 17 oligonucleotides (SEQ ID NOs: 1-17). Thismultiplex assay mixture was tested on the same 460 patient DNA samplesderived from swabs. The reactions were run using the standard qRT-PCRprotocol described above with amplification and detection performedusing a RotorGene 3000 System Real Time PCR instrument from Corbett LifeScience.

The method of the present invention provides a two step process foranalyzing results to substantively reduce false negative and falsepositive results. The first step is to separate results according tofour classifications:

1. Positive on all 3 loci=MRSA

2. Negative on Sa442=Not MRSA

3. Positive on Sa442 and negative on mecA=Not MRSA

4. Positive on Sa442, positive on mecA and negative on SCC; orfX=Go toStep Two

Based on the first step analysis, 203 samples met the criteria of thefirst classification. 202 were correctly identified as MRSA and onesample was positive on all 3 loci and yet found to be a false positive,reflecting in fact a mixture of two species, an MSSA (SCC:orfX-positiveSa442-positive mecA-negative) and a MR-CoNS (mecA-positive).

38 samples met the criteria for the second classification and all werecorrectly identified as not being MRSA. 36 samples met the criteria forclassification 3. All were correctly identified as not being MRSA exceptfor one that was found to be a false negative, probably a result of aMRSA strain with a mutated mecA gene at the site of the primers orprobe, resulting in a negative mecA reaction.

61 samples met the criteria for the fourth classification and these weresubjected to the second step analysis prescribed by the invention,namely comparison of the cycle threshold for the Sa442 and the cyclethreshold for the mecA. In 18 cases, the cycle thresholds (CT: the cycleat which samples are first detected—reflecting the concentration of thetargeted gene) for the two assays were within three CT values of oneanother, suggesting that the positive signal of mecA gene and Sa442 genewere coming from the same organism, or if it is a mixture, bothorganisms are at the same concentration. Of these 18, 16 were MRSA and 2were MSSA, however all these were declared MRSA. In 43 cases, the cycletimes for the two assays were more than three CTs, suggesting that thesesamples did not contain MRSA and instead that the positive mecAdetection reflected one specie and the positive Sa442 detectionreflected a different specie. As predicted, all 43 of these samplesproved to contain mixtures reflecting MSSA and a MR-CoNS.

The combined assay resulted in a sensitivity of 99.6%, a specificity of97.4%, a positive predictive value of 98.6% and a negative predictivevalue of 99.1% in detecting MRSA from mixed culture samples, thusdemonstrating the increased accuracy and specificity of the assays ofthe present invention over both the double and single locus assaystraditionally used to detect antibiotic-resistant bacteria such as MRSA.

Example 4 Triple Locus Assay Using Ambient Temperature-StabilizedqRT-PCR Mixture

An ambient temperature qRT-PCR reaction mix was developed containingbuffer ×1 (10 mM Tris pH 8.3, 50 mM KCl), MgCl₂, dNTPs mix, 1.5 units to2.5 units of Hot Start Thermophilic DNA polymerase and stabilizingagents. 25 microliters of mix were placed into PCR microtubes, and thehydration was reduced in each microtube by at least 50%. The microtubeswere subsequently stored at ambient temperature.

The triple locus high density multiplex qRT-PCR assay, containing 17oligonucleotides (as described in Example 3, herein above) was added tomicrotubes containing the ambient temperature qRT-PCR reaction mix. Themultiplex assay mixture was tested on 20 known patient DNA samplesplated from swabs and four control samples. The reactions were run usingthe standard qRT-PCR protocol described above with amplification anddetection performed using a RotorGene 3000 System Real Time PCRinstrument from Corbett Life Science.

The results of this assay are presented in Table 4. All 24 assaysperformed exactly as predicted, detecting the precise gene targets ofinterest. Following the two step protocol of the invention for analysisof results as described in Example 3 above, it was possible todifferentiate between samples containing MRSA and samples not containingMRSA with 100% accuracy. The results demonstrate the utility foridentification of MRSA and discrimination of MRSA from non-MRSA bacteriafrom clinical patient samples of an ambient stabilized kit using assaysof the present invention.

TABLE 4 Type Scc:orfX Sa442 Control mecA 8 MRSA + + + or − + 4 MSSA− + + or − − 4 MR-CoNS − − + or − + 1 orfX neg. MRSA − + + or − + 2MS-CoNS − − + − 2 PL − − + − 2 Template Control − − − −

Example 5 Discrimination of MRSA from MSSA, MS-CoNS and MR-CoNS

A total of 29 experimental samples containing a mixture of knownStaphylococci DNA were prepared. None of these samples containedStaphylococci DNA that included SCC:orfX genetic markers that could bedetected using the single locus assay for detection of MREJ types i, ii,iii, iv or v. SCC:orfX is also referred to herein as the “bridgingregion”.

Samples 1-4 each contained Methicillin Sensitive Staphylococcus aureus(MSSA) found positive for the Sa442 gene and negative for the mecApolynucleotide, in one of four different logarithmic concentrations.

Samples 2-8 each contained Methicillin Resistant Coagulase-negativeStaphylococci (MR-CoNS) found positive for the mecA polynucleotide andnegative for the Sa442 polynucleotide, in one of four differentlogarithmic concentrations.

Samples 9-13 contained a mixture of known Staphylococci DNA, reflectinga fixed concentration of MSSA plus one of five different logarithmicconcentration levels of MR-CoNS.

Samples 14-18 contained a mixture of known Staphylococci DNA, reflectinga fixed concentration of MR-CoNS plus one of five different logarithmicconcentration levels of MSSA.

Samples 19-22 contained known MRSA found positive for the Sa442polynucleotide and positive for the mecA polynucleotide in each of fourdifferent Log concentration levels.

Samples 23-26 duplicated samples 19-22, reflecting known MRSA foundpositive for the Sa442 gene and positive for the mecA gene in each offour different Log concentration levels.

Samples 27-29 reflected three additional known MRSA isolates foundpositive for the Sa442 polynucleotide and positive for the mecApolynucleotide all at an optimal working concentration.

Seven additional samples were prepared as controls.

Samples 30-31 reflected two different concentration levels of known MRSADNA, found positive for all three genetic loci: the Sa442polynucleotide, the mecA polynucleotide and SCC:orfX.

Samples 32-33 duplicated samples 30 and 31.

Sample 34 contained known DNA from an MR-CoNS found positive for themecA gene and negative for the Sa442 polynucleotide.

Sample 35 contained a sample of known MRSA DNA found positive for theSa442 gene, positive for the mecA gene and negative for SCC:orfX.

Sample 36 contained no DNA.

qRT-PCR amplification and detection was performed on the experimentaland control samples using the triple locus high density assay of theinvention, using the standard qRT-PCR reaction mixture, reactionprotocol and instrument described previously.

All of the experimental and control samples performed exactly asexpected, demonstrating positive or negative findings as the case wouldbe for SCC:orfX, the Sa442 polynucleotide and the mecA polynucleotide.

Twenty-one of the samples (items 9-29 and 35) were designed to reflectthe positive finding of an S. aureus-specific polynucleotide (such asSa442), the positive finding of the mecA polynucleotide, but negativefor evidence of the SCC:orfX genetic region, originated from SCC:orfXnegative MRSA (items 19-29 and 35) or mixtures of MSSA and MR-CoNS atdifferent concentrations (items 9-18). The protocol of the inventionprovides a second step analysis process for samples that are found to bepositive for an S. aureus-specific polynucleotide (such as Sa442) andpositive for the mecA gene, but negative for SCC:orfX, namely to comparethe cycle threshold of the S. aureus specific marker detection againstthe cycle time of the mecA detection in order to determine if the samplecontains MRSA or reflects a mixture of two non-MRSA species.

The second protocol step analysis process was performed on the 21experimental samples requiring the second step analysis. Detection ofthe Sa442 gene and detection of the mecA gene were found to haveoccurred within 3 CT's of each other for all samples containing MRSA.Accordingly, using the protocol of the invention, 100% of theexperimental samples containing MRSA were correctly identified.Detection of the Sa442 gene and detection of the mecA gene were found tohave accrued at an interval of 4 CT's or greater for all mixedexperimental samples containing MSSA and MR-CoNS, and not containingMRSA except the 2 samples (item 12 and 15) which contained identicalconcentration of MSSA and MR-CoNS.

In summation, the protocol of the invention was found to be 100%accurate in discriminating mixed samples containing MSSA and MR-CoNSfrom samples containing MRSA with an atypical variant SCC:orfX regionnot detectable using the single locus assay of Huletsky et al.

The results from this assay for distinguishing between different speciesare illustrated in FIGS. 2 and 3. As shown in FIG. 2, MRSA and MSSAbacteria were distinguished from one another based on detection of theproducts of amplification reactions directed to the bridging region(also referred to herein as SCC:orfX), the mecA gene region, an S.aureus-specific polynucleotide sequence (in this embodiment, the nucgene), and a human beta globin polynucleotide sequence (used as aninternal control). In the MRSA samples, all four amplification productswere detected. In contrast, in the MSSA samples, only the S.aureus-specific and the internal control human beta globinpolynucleotide were detected.

Similarly, FIG. 3 shows the results of assays in which amplificationreactions were directed to the bridging region, the mecA gene region, anS. aureus-specific polynucleotide sequence (in this embodiment, the nucgene), and a human beta globin polynucleotide sequence (used as aninternal control). In this case, for the samples containing MR-CoNS, themecA gene polynucleotide sequence and the internal controlpolynucleotide sequences were detected, whereas in samples containingMS-CoNS, only the internal control polynucleotide sequence was detected.Thus, the assays of the present invention provide the ability todistinguish among MRSA, MSSA, MR-CoNS and MS-CoNS bacteria.

Example 6 Shelf Life Stability of a QRT-PCR Reaction Mixture andMultiplex Assay

An experiment was performed to establish the performance and shelf lifestability of a multiplex assay QRT-PCR mix incorporating oligonucleotideprimers and dual fluorescent labeled oligonucleotide probes.

An ambient temperature qRT-PCR Mix was prepared as described above inExample 4.

One primer-probe set targeting human β-Hemoglobin was prepared andpurified consisting of forward and reverse primers (SEQ ID NOs: 21 and22) and a probe of with a HEX fluorescent label added to the 5′-end andBHQ1 quencher label added to the 3′-end (SEQ ID NO: 23). A secondprimer-probe set targeting the human RNase-P gene was purchases fromApplied Biosystems Inc. (TaqMan® RNase P Detection Reagents Kit, PartNumber 4316831), consisting of a forward primer, a reverse primer and aTaqMan® Probe labeled with FAM at the 5′-end and TAMRA at the 3′-end.

All four primers and two probes were added to the ambient temperatureqRT—PCR Mix prior to the hydration reduction process. These primers andprobes were also added to a second microtube containing all of thecomponents of the ambient temperature qRT—PCR Mix but stored at −20° C.to serve as a control.

After five weeks, a tube stored at room temperature containing thedehydrated PCR mix and duplex primer-probe assay was rehydrated and asecond tube containing the control mix was removed from the freezer anddefrosted. 100 ng of human DNA was added to each tube and the mixtureswere amplified in a RotorGene 6000 System Real Time PCR instrument fromCorbett Life Science according to the following protocol: First, fifteenminute hold cycle at 95° C. This was followed by fifty cycles each withthree steps. Step one was at 95° C. for fifteen seconds, step two was at55° C. for twenty seconds and step three was at 72° C. for thirtyseconds.

The first round of experimentation was performed immediately followingthe PCR reactions and was as follows: The primer-probe assay targetinghuman β-Hemoglobin yielded the following results. The sample that wasstored at −20° C. yielded a CT value of 23.30 and the sample stored atroom temperature yielded a CT value of 23.46. The primer-probe assaytargeting human RNase-P yielded the following results. The sample thatwas stored at −20° C. yielded a CT value of 25.79 and the sample storedat room temperature yielded a CT value of 26.08.

The second round of experimentation was conducted five weeks later. Theprocedure was identical to that described above, with the exception thattwo tubes stored at room temperature and one tube stored at −20° C. wereutilized in the experiment. To each of the three tubes 100 ng of humanDNA was added and each was then amplified as described above.

The results of the second round of experimentation were as follows. Theprimer-probe assay targeting human β-Hemoglobin yielded the followingresults: the sample that was stored at −20° C. yielded a CT value of25.42 and the two samples stored at room temperature yielded a CT valueof 25.46 and 26.06. The primer-probe assay targeting human RNase-Pyielded the following results: The sample that was stored at −20° C.yielded a CT value of 23.09 and the two samples stored at roomtemperature yielded a CT value of 23.50 and 23.65.

After ten weeks, the difference in effectiveness between the stabilizedmixtures of the invention left sitting out at room temperature andcomparable mixtures stored at −20° C. was less than one cycle. Theseresults are illustrated in FIG. 4. The results clearly demonstrate theutility and stability at room temperature of the PCR mixes for multiplexReal-Time PCR.

Example 7 High Density Triple Locus qRT-PCR Assay and Comparison ofCycle Thresholds Across the Triple Locus for Detecting MRSA in Singleand Mixed Bacterial Samples and Differentiating MRSA from MSSA, MR-CoNSand a Mixed Sample of MSSA & MR-CoNS

A triple locus multiplex qRT-PCR assay mixture was developed containingfour sets of primers and probes, for a total of 12 oligonucleotides (SEQID NOs: 1-2, 9-11, 15-17, 18-20 and 24). One pair of amplificationprimers and a dual labeled probe were designed to amplify and detect themecA gene (SEQ ID NOs: 9-11). A second pair of primers and a duallabeled probe were designed to amplify and detect the S. aureus-specificnuc gene (SEQ ID NOs: 18-20). A third set of primers and a dual labeledprobe were designed to amplify and detect the region of the orfX genesurrounding the insertion site for the SCCmec element in S. aureusbacteria as would amplify in an MSSA that does not contain the SCCmecelement (SEQ ID Nos: 1-2 and 24). A fourth set of primers and a duallabeled probe designed to amplify and detect human β-globin to serve asa control assay (SEQ ID NOs: 15-17).

An ambient temperature qRT-PCR reaction mix was developed containing thetriple locus multiplex qRT-PCR assay of the invention, plus buffer ×1(10 mM Tris pH 8.3, 50 mM KCl), MgCl₂, dNTPs mix, 1.5 units to 2.5 unitsof Hot Start Thermophilic DNA polymerase and stabilizing agents. 25microliters of mix were placed into PCR microtubes, and the hydrationwas reduced in each microtube by at least 50%. The microtubes weresubsequently stored at ambient temperature.

This ambient temperature multiplex assay mixture was tested with DNAsamples of known, MRSA, MSSA, MS-CoNS and MR-CoNS strains. The assay wastested on two samples of each of the following DNA samples:

1. Pure MRSA

2. Pure MSSA

3. Pure MR-CoNS

4. A mixed sample of β-globin and MS-CoNS as a control

5. A mixed sample of MSSA and MS-CoNS

6. A mixed sample of MRSA and MSSA in equal concentrations

7. A mixed sample of MRSA, MSSA & MR-CoNS in equal concentration

8. A mixed sample of MRSA and MSSA in 1:10 ratio concentrations

9. A mixed sample of MRSA and MSSA in 10:1 ratio concentrations

10. A mixed sample of MSSA and MR-CoNS in equal concentrations

11. A mixed sample of MSSA and MR-CoNS in 1:10 ratio concentrations

12. A mixed sample of MSSA and MR-CoNS in 10:1 ratio concentrations

The reactions were run using the standard qRT-PCR protocol describedabove with amplification and detection performed using a RotorGene 3000System Real Time PCR instrument from Corbett Life Science.

The method of the present invention provides a two step process foranalyzing results to substantively reduce false negative and falsepositive results. The first step is to separate results according tofive classifications (1=mecA, 2=nuc, 3=MSSA-orfX, as provided in Table2):

1. Positive on 1+2 and negative on 3=MRSA and not MSSA

2. Positive on 2+3 and negative on 1=MSSA and not MRSA

3. Positive on 1 and negative on 2+3=MR-non-S. aureus bacteria andneither MRSA nor MSSA.

4. Negative on all three and positive on the control=not MRSA, MSSA norMR-non-S. aureus bacteria

5. Positive on 1+2+3=Go to Analysis Step Two and compare the CT valuesof the different amplicons.

Based on the first step analysis, the assay performed as predicted withall 24 samples, detecting the precise gene targets of interest. Asexpected, all three of the loci were detected in all of the samplescontaining MSSA plus one or more methicillin resistant bacteria, andthese were subjected to the second step analysis prescribed by theinvention, namely comparison of the cycle thresholds (CT: the cyclewhich are considered to be positive—reflecting the concentration of thetargeted gene) for each of the three targeted genes.

As predicted, the cycle thresholds of the nuc assay and MSSA-orfX werewithin three CT's of each other for all of the mixed samples containingMSSA and not containing MRSA, suggesting that the positive signal of nucgene was coming from the same MSSA organism as the MSSA-orfX region andnot from a separate MRSA organism. Similarly, the cycle thresholds ofthe nuc assay and the MSSA-orfX were four CT's or greater of each otherfor all of the mixed samples containing MSSA and MRSA in non-equalconcentrations (samples: 8, 9), suggesting that the positive signal ofnuc gene was coming from a combination of the MSSA organism producingthe positive MSSA-orfX signal and an MRSA organism.

Following the two-step protocol of the invention for analysis ofresults, it was possible to differentiate between the samples containingMRSA and samples not containing MRSA in non-equal concentrations(samples 8, 9) with 100% accuracy. The results demonstrate the utilityfor identification of MRSA and discrimination of MRSA from non-MRSAbacteria of the triple-locus assay and protocol of the present inventionin both pure and mixed samples of unequal concentrations.

It will be appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the invention, which are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any suitable sub-combination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A kit comprising: (a) a first set of primers suitable for amplifyinga polynucleotide sequence from a mecA gene; (b) a second set of primerssuitable for amplifying a polynucleotide sequence from a bridgingregion; and (c) a third set of primers suitable for amplifying apolynucleotide sequence from a Staphylococcus aureus-specific gene,wherein said Staphylococcus aureus-specific gene is not orfX.
 2. The kitof claim 1 which further comprises dNTPs.
 3. The kit of claim 1 whichfurther comprises a DNA polymerase.
 4. The kit of claim 1 which furthercomprises a buffer.
 5. The kit of claim 1, wherein said bridging regioncomprises the right extremity junction of SCCmec and orfX.